Goldilocks conundrum explains cryoinjury in slow-cooled amphibian embryonic cells

Cryopreservation of intact fish and amphibian eggs and embryos is challenging due to the sheer size and yolk content, which prevents proper dehydration and causes lethal intracellular ice formation during cooling. Alternatively, cryopreservation of dissociated embryonic cells allows to biobank diploid genomes. However, amphibian and fish embryos have cells of varying sizes throughout the developing embryo and cell size distribution remains understudied in cryopreservation. This study examined cell size effects during cryopreservation of dissociated embryonic cells from two amphibian species. Most work used Limnodynatses peronii blastula, gastrula, and neurula cells cryopreserved with 10% dimethyl sulfoxide (DMSO) and sucrose at 0%, 1%, or 10%. Increasing sucrose concentration improved post-thaw recovery of membrane intact cells and cell concentrations, with gastrula and neurula cells having better recovery than blastula cells. An interaction amongst cryoprotectant concentration, cell size, and embryonic stage was observed. Higher sucrose improved recovery of larger cells, but reduced recovery of smaller cells. These results supported a "Goldilocks" model of cryoinjury, in which larger cells require more time to dehydrate adequately, while smaller cells are damaged by excessive dehydration and solute effects, implying that optimal cryoprotectant conditions differ by cell size. Based on post-thaw recovery, 10% DMSO + 10% sucrose was optimal and successfully applied to the cryopreservation of neurula cells from the threatened Rawlinsonia littlejohni, marking the first report of embryonic cell cryopreservation in a threatened amphibian. Our results demonstrated the importance of cell size effects on cryoinjury and their consideration in the application of cryopreservation for amphibian conservation. In brief: Since intact amphibian eggs and embryos cannot be cryopreserved, dissociated embryonic cell cryopreservation provides an alternative avenue for biobanking embryonic diploid genomes; however, cell size effects on cryoinjury is poorly understood and the developing amphibian embryo consists of different sized cells. We demonstrated that cell size influences cryopreservation outcomes by cryopreserving embryonic cells from three developmental stages under varying cryoprotectant concentrations.